Transcriptional Regulation of the Ufm1 Conjugation System in Response to Disturbance of the Endoplasmic Reticulum Homeostasis and Inhibition of Vesicle Trafficking

<div><p>Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network.</p> </div

The effect of the UPR pathways on the expression of the Ufm1 system.

<p><b>A.</b> Immunoblotting of MEF cell lysates using Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP antibodies. GAPDH was used as a loading control. Relative ratios of proteins were measured against GAPDH using Image J software. <b>B.</b> The mRNA levels of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type and Xbp-1^−/− MEF cells that were treated with ER stress-inducing agents (TG, 0.5 µM for 16 hour; TM, 10 µM for 16 hours; and BFA, 0.5 µg/ml for 16 hours). <b>C.</b> The mRNA levels of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type and PERK^−/− MEF cells that were treated with ER stress-inducing agents. The results represented mean ± SD. *p value <0.01.</p

Ufm1 is a potential target of Xbp-1.

<p><b>A.</b> The constructs of the Ufm1 promoter used for luciferase reporter assays. Human Ufm1 promoter sequence was amplified from the genome of HCT116 cells. <b>B.</b> The minimal Ufm1 promoter responded to ER stresses. Various Ufm1 promoter constructs were transfected into 293T cells that were subsequently treated with TG (0.5 µM) and TM (10 µM) for 24 hours. The promoter activity was measured by dual luciferase reporter assays (Promega). <b>C.</b> The putative Xbp-1 binding site was responsible for Xbp-1-mediated induction of Ufm1. 293T cells were transfected with indicated Ufm1 promoter constructs along with Xbp-1 expression vector. The cells were subsequently treated with TG and TM, and the promoter activity was measured by dual luciferase assays. <b>D.</b> The Ufm1 promoter activity in wild-type and Xbp-1^−/− MEFs. The Ufm1 protomer reporter was transfected into wild-type and Xbp-1^−/− MEFs, and the promoter activity was measured by dual luciferase reporter assays (Promega). The results represented mean ± SD. *p value <0.01 and **p value <0.05. <b>E.</b> CHIP assay. The Xbp-1-DNA complex was immunoprecipitated with Xbp-1 antibody, and subject to PCR using the primers specific for Ufm1 and ERdj4 promoters.</p

Knockdown of the Ufm1 system resulted in activation of UPR.

<p><b>A.</b> Up-regulation of ER chaperone proteins and CHOP in U2OS cells with knockdown of the Ufm1 system. U2OS cells were infected with lentiviral shRNAs, selected with puromycin (1.5 µg/ml). Cell lysates were collected after 6-day incubation and subject to immunoblotting with indicated antibodies. Knockdown of the Ufm1 components were confirmed by immunoblotting. <b>B.</b> Immunostaining of PDI in Uba5 and Ufc1 knockdown cells. C. Immunostaining of PDI in C53/LZAP and RCAD/Ufl1 knockdown cells. U2OS cells were subjected to immunostaining of PDI,. Knockdown of Uba5 was confirmed by Uba5 staining, while knockdown of RCAD/Ufl1 and C53/LZAP was confirmed by C53 staining. The images were acquired by Zeiss Axio Observer D1 and Axiovision software.</p

Expression of the Ufm1 system was induced by ER stress.

<p><b>A.</b> RT-PCR results of HCT116 and HepG2 cells treated with TG (1 µM for 16 hours) and TM (10 µM for 16 hours). The results represented mean ± SEM, p value <0.01 (marked by *). The inserts in Fig. 2A showed the results of Xbp-1 splicing assays. “u” indicated the unspliced form of Xbp-1, while “s” is the spliced form. <b>B.</b> Immunoblotting of the cell lysates of HCT116 and HepG2 treated with TG and TM. Specific Ufm1 conjugates were marked by arrowheads, and the non-specific 70 kD band was indicated by a star. “Long exp.” was long exposure of the blot, while “Short exp.” was short exposure of the blot in Enhanced Chemiluminescence (ECL).</p

Optimizing single irrigation scheme to improve water use efficiency by manipulating winter wheat sink-source relationships in Northern China Plain

<div><p>Improving winter wheat grain yield and water use efficiency (WUE) with minimum irrigation is very important for ensuring agricultural and ecological sustainability in the Northern China Plain (NCP). A three-year field experiment was conducted to determine how single irrigation can improve grain yield and WUE by manipulating the “sink-source” relationships. To achieve this, no-irrigation after sowing (W0) as a control, and five single irrigation treatments after sowing (75 mm of each irrigation) were established. They included irrigation at upstanding (W_U), irrigation at jointing (W_J), irrigation at booting (W_B), irrigation at anthesis (W_A) and irrigation at medium milk (W_M). Results showed that compared with no-irrigation after sowing (W0), W_U, W_J, W_B, W_A and W_M significantly improved mean grain yield by 14.1%, 19.9%, 17.9%, 11.6%, and 7.5%, respectively. W_J achieved the highest grain yield (8653.1 kg ha^-1) and WUE (20.3 kg ha^-1 mm^-1), and W_B observed the same level of grain yield and WUE as W_J. In comparison to W_U, W_J and W_B coordinated pre- and post-anthesis water use while reducing pre-anthesis and total evapotranspiration (ET). They also retained higher soil water content above 180 cm soil layers at anthesis, increased post-anthesis water use, and ultimately increased WUE. W_J and W_B optimized population quantity and individual leaf size, delayed leaf senescence, extended grain-filling duration, improved post-anthesis biomass and biomass remobilization (source supply capacity) as well as post-anthesis biomass per unit anthesis leaf area (P_ostBA-leaf ratio). W_J also optimized the allocation of assimilation, increased the spike partitioning index (SPI, spike biomass/biomass at anthesis) and grain production efficiency (GPE, the ratio of grain number to biomass at anthesis), thus improved mean sink capacity by 28.1%, 5.7%, 21.9%, and 26.7% in comparison to W0, W_U, W_A and W_M, respectively. Compared with W_A and W_M, W_J and W_B also increased sink capacity, post-anthesis biomass and biomass remobilization. These results demonstrated that single irrigation at jointing or booting could improve grain yield and WUE via coordinating the “source-sink” relationships with the high sink capacity and source supply capacity. Therefore, we propose that under adequate soil moisture conditions before sowing, single irrigation scheme from jointing to booting with 75 mm irrigation amount is the optimal minimum irrigation practice for wheat production in this region.</p></div

Optimizing single irrigation scheme to improve water use efficiency by manipulating winter wheat sink-source relationships in Northern China Plain - Fig 3

<p><b>Leaf length (top), width (middle) and area (bottom) of the flag (a, d), second (b, e) and third leaf (c, f) at anthesis under six treatments in the 2015–2016 growing season.</b> Box boundaries indicate upper and lower quartiles, whisker caps indicate maximum and minimum value, black solid horizontal lines indicate medians and solid dots indicate mean value.</p

Optimizing single irrigation scheme to improve water use efficiency by manipulating winter wheat sink-source relationships in Northern China Plain - Fig 7

<p><b>Sink capacity under six treatments in the 2013–2014 (a), 2014–2015(b) and 2015–2016 (c) growing seasons.</b> Different letters in the figure indicate statistical differences among treatments (LSD_{<i>P</i><0.05}).</p

Biomass at anthesis and maturity, post-anthesis biomass and biomass remobilization under six treatments in the 2013–2014, 2014–2015 and 2015–2016 growing seasons.

<p>Different letters in the figure indicate statistical differences among treatments (LSD_{<i>P</i><0.05}).</p

Optimizing single irrigation scheme to improve water use efficiency by manipulating winter wheat sink-source relationships in Northern China Plain - Fig 4

<p><b>Leaf area index (LAI) of top three leaves and total green leaves at anthesis under six treatments in the 2013–2014 (a), 2014–2015 (b) and 2015–2016 (c) growing seasons.</b> Different letters in the figure indicate statistical differences among treatments (LSD_{<i>P</i><0.05}).</p